Type V collagen induces apoptosis of 8701-BC breast cancer cells and enhances m-calpain expression
© The Author(s) 2000
Received: 12 January 2000
Published: 1 June 2000
We previously reported that ductal infiltrating carcinomas (DIC) of the human breast display profound modifications of the stromal architecture, associated with anomalous collagen composition. The major alterations observed in the interstitial collagen were an abnormal ratio between type I and type III collagens, the appearence of an onco-foetal form of collagen (OF/LB) and a relative increase of type V collagen content. Biological assays performed by culturing a DIC-derived cell line (8701-BC) onto type V collagen substrate demonstrated that the latter was able to restrain cell growth and to inhibit cell motility and invasion "in vitro", differently from what found with other collagen species tested.
To search for molecular mechanisms underlying the observed inhibitory effect of collagen type V on breast cancer cells. As a reference model, we used culture substrates prepared with type IV collagen, which represents the physiological substrate for cells of epithelial origin.
Apoptosis was studied by both fluorescence microscopical analyses of cell viability and DNA fragmentation assays in preparations of 8701-BC cells grown onto either type V and type IV collagen. Differences in gene expression following cell adhesion onto the two substrates were analyzed by a "differential display" PCR (DD-PCR) technique and Western blot.
In this paper we demonstrate that the inhibitory effect exerted by type V collagen is consistently associated with the switching-on of a death program by a significant proportion of the cell culture, concomitant with the formation of cohesive cell islands displaying a progressive decrease of cell spreading. DD-PCR and Western blot assays demonstrated a consistent association of type V collagen-promoted apoptosis with the up-regulation of the large subunit of m-calpain (L-mC) at both mRNA and protein level. Cell exposure to calpain inibitor I decreased the amount of DNA fragmentation by 30%.
The present data substantiate our previous postulates that in cases of breast DIC the zonal increase of type V collagen contribute to the assembly of a "non-permissive" micro-environment for tumor cells, antagonist to other local permissive substrates. It is therefore conceivable that the spatio-temporal derangement of stromal components may actively modulate neoplastic cell behavior and metastatic propensity, thus contributing to the selection and development of more or less malignant tumor phenotypes.
The interdynamic space-temporal interactions of given cells with their extracellular matrix (ECM) are known to drive morphogenesis during development and to maintain the normal tissue architecture and functions in adult organs. Cellular behaviors that are modulated by (or associated to) cell-matrix interactions include growth [1,2], differentiation [3,4,5], apoptosis , motility [7,8,9], signal transduction and gene expression [10,11,12,13,14], to quote among the major effects. Local disruption of ECM after pathologic events, may result in selective reprogrammed cell behavior, through a cascade of signals difficult to predict a priori.
Some previous data obtained in one of our laboratories have shown that in cases of the ductal infiltrating carcinoma (DIC), a highly malignant tumor of the human breast, the stromal ECM undergoes to extensive regional remodelling with enhanced deposition of collagen components, mostly modified with respect to the normal counterpart [15,16]. The major aspects were the over-deposition of type V collagen normally expressed at very low levels in adult tissues, and the appearence an onco-foetal form of interstitial collagen (OF/LB) [17,18,19]. Biological assays performed with a neoplastic cell line (8701-BC), isolated from a primary DIC , demonstrated that different collagen substrates were able to promote distinct behaviors of the cell population: in particular, the onco-foetal collagen enhanced cell proliferation and migration and promoted invasivity both "in vitro" and "in vivo", whilst type V collagen exerted an antagonistic effect, restraining both cell growth and "in vitro" invasivity [2,8,21,22,23,24].
In consideration of the potentially-relevant implications of restrictive forces on tumor progression, we set out experiments to understand how this inhibitory effect of collagen type V is exerted on cancer cells. As a reference model, we used culture substrates prepared with type IV collagen, which represents the physiological substrate for cells of epithelial origin. First, to address the question whether type V collagen was able to promote programmed cell death, we performed fluorescence microscopical analyses of cell viability and DNA fragmentation assays, both by gel electrophoresis and centrifugal sedimentation. Secondly, we investigated the possibility of some transcriptional specificity induced by type V collagen substrates on 8701-BC cells, by applying the technique of the "differential display"-polymerase-chain-reaction (DD-PCR) described by Sokolov and Prockop  which allows the identification of cDNas from differentially-expressed genes by analysis of their internal sequences after reverse transcription with random hexamers and amplification in the presence of arbitrary primers.
By this experimental approach, here we demonstrate that type V collagen is able to promote apoptosis in a large fraction of 8701-BC cells, and that the apoptotic process is associated with a marked increase of expression and production of the large subunit of m-calpain (L-mC; EC 220.127.116.11), a neutral Ca++-dependent non-lysosomal enzyme belonging to the cystein protease family ([26,27,28] for reviews).
MATERIALS AND METHODS
8701-BC cells were routinely cultured in RPMI 1640 medium supplemented with 10% foetal calf serum and antibiotics. Collagen type IV and V were purchased from Sigma (St.Louis, MO/USA). Collagens were dissolved in 0.5 M acetic acid, sterilized with chloroform as already reported in Luparello et al. , plated in 25 cm2 flasks at the concentration of 10 μg/cm2 for 48 h and exhaustively neutralized with PBS just before cell culturing. 8701-BC cells, detached with 0.1% EDTA, were plated in substrate-coated flasks at the concentration of 3.5 x 104/cm2in serum-free conditions and allowed to grow for 48 h.
A mixture of acridine orange and ethidium bromide (both purchased from Sigma and dissolved at 2 μg/ml of PBS) was added to the culture medium of unfixed 8701-BC cells grown for 48 h onto either type IV or type V collagen substrate, and the cells viewed immediately under either FITC (for acridine orange) or TRITC (for ethidium bromide) fluorescence  and photographed.
DNA fragmentation assays
The occurrence of DNA fragmentation was evaluated electrophoretically as described by Navarro et al.  with slight modifications, and its extent checked by centrifugal sedimentation as reported by Duke and Cohen .
For the electrophoretic assay, 8701-BC cells were plated onto either type IV collagen- or type V collagen-coated 6-well dishes, at the concentration of 6 x 105 (for type IV collagen) and 1.2 x 106cells/well (for type V collagen) and grown for 48 h in serum-free medium. At the end of the incubation, cells were scraped from the plates, lysed in 100 μl of 45 mM Tris-borate, pH 8, containing 0.25% Nonidet P-40 substitute and 10 mM EDTA, and treated for 1 h at 50°C with 1 mg RNase A/ml (Fluka, Buchs/CH) and then for 1 h at 50°C with 1 mg Proteinase K/ml (Fluka). Samples were mixed with loading buffer (10 mM EDTA, pH 8, plus 0.25% bromophenol blue, 40% sucrose and 1% low melting point-agarose) and, in order to improve band resolution, they were submitted to voltage gradient gel electrophoresis (VGGE; [32,33]) in ethidium bromide-containing 1.2% agarose gel with TAE buffer for 5 h at 60 V.
For the quantitative evaluation of DNA fragmentation, 8701-BC cells were plated at the concentration of 1 × 105 cells/well onto either type IV collagen- or type V collagen-coated 4-well dishes in serum-free conditions and allowed to adhere onto the substrates for 24 h. Subsequently, a set of cells was incubated with 2 μCi/ml [methyl-3H] thymidine ([3H]TdR), for 18 h, whilst in parallel preparations, the 18 h-treatment with [3H]TdR was performed after additonal 24 h (i.e. at 48 h from seeding). Cells were then exhaustively washed with RPMI 1640 medium pre-warmed to 37°C and lysed with TTE solution (10 mM Tris-HCl, pH 7.4, containing 0.2% Triton X-100 and 1 mM EDTA); the fragmented DNA was then separated from intact chromatin by centrifugation. The radioactivity present in both the supernatant and the pellet, the latter resuspended in 1% SDS, was evaluated by liquid scintillation counting. Percent DNA fragmentation was calculated as [(c.p.m in the supernatant)/(c.p.m. in the supernatant plus c.p.m. in the pellet)]. Data are presented as mean ± s.e.m. of quadruplicate experiments. A Student's t-test was used and p < 0.05 was taken as the minimal level of statistical significance of the differences between samples and controls. the dna fragmentation assay following calpain inhibition followed essentially the same protocol with the exception that either 20 μm calpain inhibitor i (N-acetyl-Leu-Leu-norleucinal purchased by Boehringer; ) in DMSO or DMSO vector only were added to cell plating medium.
Messenger RNA extraction and reverse transcription
Isolation of polyA+ mRNA from monolayers of 8701-BC cells grown for 48 h onto either type IV or V collagen substrate was carried out with oligo-dT25tailed magnetic beads using the mRNA DIRECT kit purchased from Dynal (Oslo/N). The protocol for the latter included the preparation of crude cell lysate, annealing of polyadenylated mRNA to the beads, washing of the suspension and elution of purified transcripts. Samples of 0.1-0.5 μg of mRNA were submitted to cDNA synthesis in the presence of random 6-mer primers, using the SuperScript Preamplification System (Gibco), according to manufacturer's instructions.
Differential Display-Polymerase Chain Reaction (DD-PCR) and Sequencing
For differential expression analysis, DD-PCR experiments were performed using the arbitrary 10-mer primers designed by Sokolov and Prockop , i.e. BS52 (5'-CAAGCGAGGT-3'), BS54 (5'-AACGCGCAAC-3'), BS55 (5'-GTGGAAGCGT-3') and BS57 (5'-GGAAGCAGCT-3'), in combinations of two. The PCR amplification was carried out using 25 pmoles of each of two primers, 1-2 μl of the cDNA template and 3.6 U of AmpliTaq DNA Polymerase, Stoffel fragment (Perkin Elmer, USA), in 50 μl of the appropriate reaction mixture. The thermal cycle used was a denaturation step of 94.5°C for 3 min., followed by 45 cycles of 94.5°C for 1 min., 34°C for 1 min., 72°C for 1 min. and a final extension of the product for 10 min. at 72°C. PCR products were analysed by 6% non-denaturing polyacrylamide gel electrophoresis, followed by silver staining [35,36]. PBR322 plasmid digested with HinfI was used as size marker (154, 220/221, 298, 344, 396, 506, 517 and 1,631 bp fragments). The cDNA fragment of interest was excised from the gel and subjected to several repetitions of amplification and electrophoresis until purity. Direct dideoxy-sequencing of both strands of the PCR product, purified with the High Pure PCR Product Purification Kit (Boehringer), was carried out with Sequenase (Amersham, UK) in the presence of either BS52 or BS57 primer and 33P-labelled dATP. DNA sequence similarity was searched with the BLAST algorithm  available on-line at http:\\www.ncbi.nlm.nih.gov.
Non-competitive semi-quantitative PCR was performed at low cycle number  using 50 μM of the primers for L-mC (accession nr. M23254), i.e. 5'-CAAAAACTTCTTCCTGACGAATCG-3' (sense) and 5'-CCAGACCTGTCAACGTCGATT-3' (antisense), designed with the Primer Selection software available on-line at http://alces.med.umn.edu, and specific for a 512 bp sequence from bases 1477-1989 of the L-mC coding sequence . Cycle profile was 94°C for 2 min., followed by 18 cycles of 94°C for 30 sec., 47°C for 45 sec., 72°C for 1 min (5 min. during the last cycle). Primewax (Biometra) was added for "hot start". GAPDH was amplified in parallel as described by Southby et al. . PCR products and size marker (PBR322 plasmid/HinfI) were analysed by 6% PAGE and visualized at 254 nm transillumination after staining with SYBR Green I. SigmaGel 1.0 software was utilized for the evaluation of pixel intensity of the fluorescent bands. A diagnostic restriction test was designed using the WWW tacg program, available at http:\\hornet.bio.uci.edu; according to the data obtained, the amplification product of L-mC cDNA, purified by High Pure PCR Product Purification Kit (Boehringer), was digested with excess PstI enzyme and the digest analyzed by PAGE as described.
Protein extraction and Western blot
Cells were plated in collagen-coated flasks in serum-free conditions and allowed to grow for 48 h, as already reported. Cell lysis was performed with 20 mM Tris-HCl, pH 7.4, containing 2% SDS, 5 mM EGTA, 5 mM EDTA, 0.5 mM PMSF, 5 μg leupeptin/ml and 10 μg aprotinin/ml; total proteins were extracted and TCA precipitated according to Wang et al. . Measured aliquots of the extracted proteins were subjected to SDS-PAGE (7.5% acrylamide). Western-blot was performed with anti-L-mC antibody from rabbit (1:1,000; Swant, Bellinzona/CH), and the reaction revealed by horseradish peroxidase-conjugated anti-rabbit IgG (1 U/ml; Boehringer) and staining with diaminobenzidine according to . The evaluation of the pixel intensity of the bands was done by automated scanning using SigmaGel 1.0 software. Beta-actin was immunostained in parallel to confirm the loading of the proper amount of material.
Apoptosis of 8701-BC cells cultured onto type V collagen
Up-regulation of L-mC in 8701-BC cells cultured onto type V collagen
Inhibition of calpain and reduction of the extent of DNA fragmentation in 8701-BC cells grown onto type V collagen
To check whether calpain played a role in the apoptosis induced on 8701-BC cells by type V collagen, a cell-permeant inhibitor of calpain, calpain inhibitor I , was used at the concentration recommended by Squier et al. .
Type V collagen, a minor component of the multigenic collagen family, was originally detected in foetal membranes  and subsequently found, as a minor fraction of the total collagen, in several embryonic and adult tissues, where it occurs in a pericellular localization [44,45]. However, although the molecular and structural characteristics of type V collagen have been thoroughly investigated (see ), its biological role remains uncertain.
Some authors demonstrated that in mature tissues type V collagen molecules are buried within the major collagen fibrils [47,48] and therefore, in normal situations the epitopes for cell adhesion are not readily available to cells. Conversely, during tissue remodelling or in tumor invasion, enhanced deposition of collagen type V has been observed [19,49,50], and it is conceivable that it could become transiently available for cell adhesion, driving some specific cellular functions. However, the data so far available show that the cellular responses driven by this collagen appear inconsistent, suggesting differences among the model systems examined (e.g. tissues and cell types). For example, cell adhesion-promoting activity has been observed for normal smooth muscle cells [51,52], chinese hamster ovary cells , and some tumor cells ; conversely, anti-adhesive and anti-proliferative effects have been observed on endothelial cells [55,56,57], vascular smooth muscle cells  and some epithelial cells , besides the 8701-BC cancer cell line .
Present results confirm our previous observations on the "in vitro" inhibitory effect of type V collagen on cell proliferation, and offer an explanation of the biological mechanisms involved. Indeed, the decrease of the expected growth rate in a given cell population may be due to two possible responses: a slow-down of the duplication rates or an increase rate of programmed cell death. The data presented here clearly support the second hypothesis. As it is known, apoptosis is a cell death program that produces specific morphological and biochemical modification, including loss of adherence to the substrate, cell shrinking in volume and chromatin condensation and separation into discrete masses, that produce a ladder-like pattern when the fragmented DNA is electrophoresed through agarose gels . All the above-mentioned phenomenoma have been shown to occur in a large percentage of cells cultured onto type V collagen substrate.
Concerning the cell pathways involved in the apoptotic process, the knowledge is still fragmentary, but it is known that it invariably requires the activation of proteolytic cascades that are not yet well-defined. Cystein proteases, a family of non-lysosomal neutral proteases whose major members are calpain and caspases, have been considered as important intermediates in this cascade . Some authors suggested that the proteolytic cascade is initiated after upstream caspase activation , others that calpains and the proteasome (a multicatalytic enzymatic complex) function synergistically downstream of caspases . Concerning calpain substrates, cytoskeletal components (e.g. spectrin, fodrin, actin), membrane-linked proteins (e.g. Ca++ pump) and nuclear/cytosolic proteins , including cyclin D1 , have been recognised in a number of "in vitro" assays. Villa et al.  have suggested that the correlation between calpain-driven apoptosis and cytoskeleton might be ascribed to the efficacy of calpain to release DNase I from actin microfilaments, thus becoming more available for DNA fragmentation. Interestingly, as a support to this hypothesis in our model system, previous fluorescence microscopical analysis of actin in 8701-BC cells plated on type V collagen revealed the lack of a well-organized microfilament array , which was instead prominently present in cells cultured onto other collagen substrates [8,21].
Due to the importance of the apoptotic process in tissue homeostasis and in cancer biology and therapy, in the last few years a multiplicity of substances has been investigated as possible inducer/blockers of apoptosis (e.g. ), but, to our knowledge, this is the first documented indication that an endogenous tissue component, like type V collagen, is able to promote apoptosis in cancer cells. The expression of other components required for the apoptotic activation following 8701-BC adhesion onto type V collagen is currently under investigation
The present data substantiate our previous postulates that in cases of breast DIC the zonal increase of type V collagen contribute to the assembly of a "non-permissive" micro-environment for tumor cells, antagonist to other local permissive substrates [2,8,21,23,24]. It is therefore conceivable that the spatio-temporal derangement of stromal components may actively modulate neoplastic cell behavior and metastatic propensity, thus contributing to the selection and development of more or less malignant tumor phenotypes.
This work was suppported by A.I.R.C. and M.U.R.S.T. (R.S. ex 60% and 40%)
- Chou JL, Shen ZX, Stolfi RL, Martin DS, Waxman S: Effects of extracellular matrix on the growth and casein gene expression of primary mouse mammary tumor cells in vitro . Cancer Res. 1984, 49: 5371-5376.Google Scholar
- Luparello C, Schillaci R, Pucci-Minafra I, Minafra S: Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen. Eur J Cancer. 1990, 26: 231-240.View ArticlePubMedGoogle Scholar
- Adams JC, Watt FM: Regulation of development and differentiation by the extracellular matrix. Development. 1993, 117: 1183-1198.PubMedGoogle Scholar
- Hay ED: Extracellular matrix alters epithelial differentiation. Curr Opin Cell. Biol. 1993, 5: 1029-1035.View ArticlePubMedGoogle Scholar
- Streuli CH: Extracellular matrix and gene expression in mammary epithelium. Semin Cell Biol. 1993, 4: 203-212.View ArticlePubMedGoogle Scholar
- Boudreau N, Werb Z, Bissell MJ: Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. Proc Natl Acad Sci. USA. 1996, 93: 3509-3513.View ArticlePubMedPubMed CentralGoogle Scholar
- Klemke R, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA: Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol. 1997, 137: 481-492.View ArticlePubMedPubMed CentralGoogle Scholar
- Luparello C, Sheterline P, Pucci-Minafra I, Minafra S: A comparison of spreading and motility behaviour of 8701-BC breast carcinoma cells on type I, I-trimer and type V collagen substrates. J Cell Sci. 1991, 100: 179-185.PubMedGoogle Scholar
- Zetter BR, Brightman SE: Cell motility and the extracellular matrix. Curr Opin Cell Biol. 1990, 2: 850-856.View ArticlePubMedGoogle Scholar
- Juliano RL, Haskill S: Signal transduction from the extracellular matrix. J Cell Biol. 1993, 120: 577-586.View ArticlePubMedGoogle Scholar
- Minafra S, Giambelluca C, Andriolo M, Pucci-Minafra I: Cell-cell and cell-collagen interactions influence gelatinase production by human breast carcinoma cell line 8701-BC. Int J Cancer. 1995, 62: 1-7.View ArticleGoogle Scholar
- Roskelley CD, Srebrow A, Bissell MJ: A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Curr Opin Cell Biol. 1995, 7: 736-747.View ArticlePubMedPubMed CentralGoogle Scholar
- Danen E, Lafrenie R, Miyamoto S, Yamada K: Integrin signaling: cytoskeletal complexes, MAP kinase activation, and regulation of gene expression. Cell Adhes Commun. 1998, 6: 217-224.View ArticlePubMedGoogle Scholar
- Lelièvre S, Weaver V, Nickerson J, et al: Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus. Proc Natl Acad Sci USA. 1998, 95: 14711-14716.View ArticlePubMedPubMed CentralGoogle Scholar
- Minafra S, Pucci-Minafra I, Tomasino RM, Sciarrino S: Collagen composition in the ductal infiltrating carcinoma of human breast. Cell Biol Int Rep. 1984, 8: 79-85.View ArticlePubMedGoogle Scholar
- Pucci-Minafra I, Luparello C, Sciarrino S, Tomasino RM, Minafra S: Quantitative determination of collagen types present in the ductal Infiltrating carcinoma of human mammary gland. Cell Biol Int Rep. 1985, 9: 291-296.View ArticlePubMedGoogle Scholar
- Pucci-Minafra I, Luparello C, Andriolo M, Basiricò L, Aquino A, Minafra S: A new form of tumor and foetal collagen with laminin-binding property. Biochemistry. 1993, 32: 7421-7427.View ArticlePubMedGoogle Scholar
- Pucci-Minafra I, Andriolo M, Basiricò L, et al: Onco-fetal/laminin-binding collagen from colon carcinoma: detection of new sequences. Biochem Biophys Res Commun. 1995, 207: 852-859. 10.1006/bbrc.1995.1264.View ArticleGoogle Scholar
- Luparello C, Rizzo CP, Schillaci R, Pucci-Minafra I: Fractionation of type V collagen from carcinomatous and dysplasic breast in the presence of alkaline potassium chloride. Analyt Biochem. 1988, 169: 26-32.View ArticlePubMedGoogle Scholar
- Minafra S, Morello V, Glorioso F, et al: A new cell line (8701-BC) from primary ductal infiltrating carcinoma of human breast. Br J Cancer. 1989, 60: 185-192.View ArticlePubMedPubMed CentralGoogle Scholar
- Schillaci R, Luparello C, Minafra S: Type I and I-trimer collagens as substrates for breast carcinoma cells in culture. Effect on growth rate, morphological appearence and actin organization. Eur J Cell Biol. 1989, 48: 135-141.PubMedGoogle Scholar
- Pucci-Minafra I , Luparello C: Type V/type I collagen interactions "in vitro" and growth-inhibitory effect of hybrid substrates on 8701-BC carcinoma cells. J Submicrosc Cytol Pathol. 1991, 23: 67-74.PubMedGoogle Scholar
- Luparello C: Adhesion to type V collagen and cloning efficiency in agar of 8701-BC breast cancer cells. Eur J Cancer. 1994, 30A: 1400-1401.View ArticlePubMedGoogle Scholar
- Pucci-Minafra I, Luparello C, Aquino A, et al: OF/LB collagen promotes chemoinvasion of breast cancer cells and directes epithelial cell migration into granulation tissue of experimental dermal wounds. Int J Oncol. 1995, 6: 1015-1020.Google Scholar
- Sokolov BP, Prockop DJ: A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucl Acid Res. 1994, 19: 4009-4015.View ArticleGoogle Scholar
- Johnson GVW, Guttmann RP: Calpains: intact and active?. Bioessays. 1997, 19: 1011-1018.View ArticlePubMedGoogle Scholar
- Carafoli E, Molinari M: Calpain: a protease in search of a function?. Biochem Biophys Res Commun. 1998, 247: 193-203. 10.1006/bbrc.1998.8378.View ArticlePubMedGoogle Scholar
- Ono Y, Sorimachi H, Suzuki K: Structure and physiology of calpain, an enigmatic protease. Biochem Biophys Res Commun. 1998, 245: 289-294. 10.1006/bbrc.1998.8085.View ArticlePubMedGoogle Scholar
- Edwards SN, Tolkovsky : Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal. J Cell Biol. 1994, 124: 537-546.View ArticlePubMedGoogle Scholar
- Navarro JN, Olmo N, Lòpez-Conejo MT, Lizarbe MA: Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5'-nucleotidase activity. Eur J Clin Invest. 1997, 27: 620-628.View ArticlePubMedGoogle Scholar
- Duke RC, Cohen JJ: Morphological and biochemical assays of apoptosis. In Current Protocols in Immunology. Edited by Coogan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W. New York: Green Publishing and Wiley-Interscience. 1992, 3.17: 1-3.Google Scholar
- Barbieri R, Duro G, Izzo V: Enhanced hybridization labelling signals in Southern blotted DNAs fractionated with voltage gradient gel electrophoresis. Electrophoresis. 1998, 19: 643-645.View ArticlePubMedGoogle Scholar
- Asaro MR, Izzo V, Barbieri R: Modified apparatus for voltage gradient gel electrophoresis. J Chromatogr. 1999,Google Scholar
- Squier MKT, Miller ACK, Malkinson AM, Cohen JJ: Calpain activation in apoptosis. J Cell Physiol. 1994, 159: 229-237.View ArticlePubMedGoogle Scholar
- Caetano-Anollés G, Bassam BJ, Gresshoff PM: Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides. Mol Gen Genet. 1992, 235: 157-165.View ArticlePubMedGoogle Scholar
- Doss RP: Differential display without radioactivity - A modified procedure. BioTechniques. 1996, 21: 408-412.PubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1006/jmbi.1990.9999.View ArticlePubMedGoogle Scholar
- Gause WC, Adamovicz J: The use of the PCR to quantitate gene expression. PCR Meth Appl. 1994, 3: S123-S135.View ArticleGoogle Scholar
- Imajoh S, Aoki K, Ohno S, et al: Molecular cloning of the cDNA for the large subunit of the high-Ca++-requiring form of human Ca++-activated neutral protease. Biochemistry. 1988, 27: 8122-8128.View ArticlePubMedGoogle Scholar
- Southby J, O'Keefe LM, Martin TJ, Gillespie MT: Alternative promoter usage and mRNA splicing pathways for parathyroid hormone-related protein in normal tissues and tumours. Br J Cancer. 1995, 72: 702-707.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang KKW, Posner A, Hajimohammadreza I: Total protein extraction from cultured cells for use in electrophoresis and western blotting. BioTechniques. 1996, 20: 662-668.PubMedGoogle Scholar
- Sasaki T, Kishi M, Saito M, et al: Inhibitory effect of di- and tripeptidyl aldehydes on calpains and calpastatins. J Enzyme Inhib. 1990, 3: 195-201.View ArticlePubMedGoogle Scholar
- Burgeson RE, El Adli FA, Kaitila II, Hollister DW: Fetal membrane collagens: identification of two new collagen α chains. Proc Natl Acad Sci USA. 1976, 73: 2579-2583.View ArticlePubMedPubMed CentralGoogle Scholar
- Gay S, Rhodes RK, Gay RE, Miller EJ: Collagen molecules comprised of α1 (V)-chains (B-chains): an apparent localization in the exocytoskeleton. Collagen Rel Res. 1981, 1: 53-58.View ArticleGoogle Scholar
- Modesti A, Kalebic T, Scarpa S, et al: Type V collagen in human amnion is a 12 nm-fibrillar component of the pericellular interstitium. Eur J Cell Bio. 1984, 35: 246-255.Google Scholar
- Fichard A, Kleman JP, Ruggiero F: Another look at collagen V and XI molecules. Matrix Biol. 1994, 14: 515-531.View ArticleGoogle Scholar
- von Der Mark K, Ocalan M: Immunofluorescent localization of type V collagen in the chick embryo with monoclonal antibodies. Collagen Rel Res. 1982, 2: 541-555.View ArticleGoogle Scholar
- Linsenmayer TF, Gibney E, Igoe F, et al: Type V collagen: molecular structure and fibrillar organization of the chicken α(1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis. J Cell Biol. 1993, 121: 1181-1189.View ArticlePubMedGoogle Scholar
- Barsky S, Rao C, Grotendorst G, Liotta L: Increased content of type V collagen in desmoplasia of human breast carcinoma. Am J Pathol. 1982, 108: 276-283.PubMedPubMed CentralGoogle Scholar
- Marian B, Danner MW: Skin tumor promotion is associated with increased type V collagen content in the dermis. Carcinogenesis. 1987, 8: 151-154.View ArticlePubMedGoogle Scholar
- Grotendorst GR, Seppä HE, Kleinman HK, Martin GR: Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor. Proc Natl Acad Sci USA. 1981, 78: 3669-3672.View ArticlePubMedPubMed CentralGoogle Scholar
- Leushner JR, Haust MD: Glycoproteins on the surface of smooth muscle cells involved in their interaction with type V collagen. Can J Biochem Cell Biol. 1985, 63: 1176-1182.View ArticlePubMedGoogle Scholar
- LeBaron RG, Höök A, Esko JD, Gay S, Höök M: Binding of heparan sulfate to type V collagen. A mechanism of cell-substrate adhesion. . J Biol Chem. 1989, 264: 7950-7956.PubMedGoogle Scholar
- Ruggiero F, Champliaud MF, Garrone R, Aumailley M: Interactions between cells and collagen V molecules or single chains involve distinct mechanisms. Exp Cell Res. 1994, 210: 215-223. 10.1006/excr.1994.1032.View ArticlePubMedGoogle Scholar
- Fukuda K, Koshihara Y, Oda H, Ohyama M, Ooyama T: Type V collagen selectively inhibits human endothelial cell proliferation. Biochem Biophys Res Commun. 1988, 151: 1060-1068.View ArticlePubMedGoogle Scholar
- Madri JA, Williams SK: Capillary endothelial cell cultures: phenotypic modulation by matrix components. J Cell Biol. 1983, 97: 153-165.View ArticlePubMedGoogle Scholar
- Ziats N, Anderson JM: Human vascular endothelial cell attachment and growth inhibition by type V collagen. J Vasc Surg. 1993, 17: 710-718.View ArticlePubMedGoogle Scholar
- Underwood PA, Bean PA, Whitelock JM: Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen. Atherosclerosis. 1998, 141: 141-152. 10.1016/S0021-9150(98)00164-6.View ArticlePubMedGoogle Scholar
- Parekh T, Wang X, Makri-Werzen DM, Greenspan DS, Newman MJ: Type V collagen is an epithelial cell cycle inhibitor that is induced by and mimics the effects of transforming growth factor ß1. Cell Growth Differ. 1998, 9: 423-433.PubMedGoogle Scholar
- Martin SJ, Green DR, Cotter TG: Dicing with death: dissecting the components of the apoptosis machinery. Trends Biochem Sci. 1994, 19: 26-30.View ArticlePubMedGoogle Scholar
- Kohli V, Madden JF, Bentley RC, Clavien PA: Calpain mediates ischemic injury of the liver through modulation of apoptosis and necrosis. Gastroenterology. 1999, 116: 168-78.View ArticlePubMedGoogle Scholar
- Waterhouse NJ, Finucane DM, Green DR, et al: Calpain activation is upstream of caspases in radiation-induced apoptosis. Cell Death Differ. 1998, 5: 1051-1061. 10.1038/sj/cdd/4400425.View ArticlePubMedGoogle Scholar
- Knepper NB, Savill J, Brown SB: Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases. J Biol Chem. 1998, 273: 30530-30536.View ArticleGoogle Scholar
- Choi YH, Lee SJ, Nguyen P, et al: Regulation of cyclin D1 by calpain protease. J Biol Chem. 1997, 272: 28479-28484.View ArticlePubMedGoogle Scholar
- Villa PG, Henzel WJ, Sensenbrenner M, Henderson CE, Pettmann B: Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis. J Cell Sci. 1998, 111: 713-722.PubMedGoogle Scholar
- Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, Kline K: Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutrition Cancer. 1999, 33: 26-32.View ArticlePubMedGoogle Scholar